EXCEED THE SPACE PROVIDED. ABSTRACT The primary objective of the proposed research is to understand the mechanisms by which distinct combinations of alternatively spliced protocadherin mRNAs are expressed in individualneurons in the brain. This objective will be approached at the levels of transcription and pre-mRNA splicing. Transcription mechanisms will be addressed by integrating reporter genes downstream from individual protocadherin promoters and then studying their cell-specific pattern of expression in the brain. The regulatory sequences required for the expression of individual protocadherin isoforms will then be identified by a combination of chromatin digestion experiments and inter-species genomic DNA sequence comparisons. Once identified, these sequences will be altered by deletion or mutation in bacterial artificial chromosomes (BACs), and the effects of these deletions tested in embryonic stem cells and in transgenic mice. The pre-mRNA splicing experiments will be directed towards determining whether protocadherin mRNAs are produced by a cis-or fr[unreadable]rc.s-splicing mechanism. This will be accomplished by direct analysis of pre-mRNA splicing intermediates and by various manipulations of BACs aimed at disrupting transcription throughout the gene clusters. The effects of these manipulations will be tested by introducing the BACs into mouse ES cells. If successful, the proposed experiments would significantly advance our understandingof the mechanisms for generating protein diversity by combinatorial activation of promoters and alternative pre-mRNA splicing. In addition, they would provide important insights into understanding the molecular basis of specific neuronal connectivity in the brain. An understanding of these basic processes might ultimately contribute to the understanding and treatment of behavioral and cognitive disorders.